A pyruvate dehydrogenase complex, utilized for quantitative determination of pyruvate acid, etc., finds various utilities because pyruvic acid is an intermediate for many reaction systems.
The pyruvate dehydrogenase complex as a whole is an enzyme that catalyzes oxidative decarboxylation of pyruvic acid in the presence of TPP (thiamin pyrophosphate) or lipoamide as a coenzyme, as follows: EQU Pyruvic acid+NAD.sup.+ +CoA.fwdarw.Acetyl-CoA+CO.sub.2 +NADH+H.sup.+
This enzyme is actually an enzyme complex of molecular weight of several millions consisting of 3 types of enzyme proteins (E.sub.1, E.sub.2 and E.sub.3), each of which catalyzes one of the following reaction steps. Out of the complex, the present invention relates to the E1 protein (pyruvate dehydrogenase). EQU Pyruvic acid+E.sub.1 -TPP.fwdarw.E.sub.1 -TPP-CHOH-CH.sub.3 +CO.sub.2
{E.sub.1 : pyruvate dehydrogenase} EQU E.sub.1 -TPP-CHOH-CH.sub.3 +E.sub.2 -LipS.sub.2 +CoA.fwdarw.E.sub.1 -TPP+acetyl-CoA+E.sub.2 -Lip(SH).sub.2 PA1 {E.sub.2 : dihydrolipoamide acetyltransferase} EQU E.sub.3 EQU E.sub.2 -Lip(SH).sub.2 +NAD.sup.+ .rarw..fwdarw.E.sub.2 -LipS.sub.2 +NADH+H.sup.+ PA1 {E.sub.3 : lipoamide dehydrogenase}
{in the above reactions, TPP is thiamin pyrophosphate, LipS.sub.2 is lipoic acid, and Lip(SH).sub.2 is dihydrolipoic acid.}
As E.sub.1 protein of wild-type pyruvate dehydrogenase complex and a nucleotide sequence coding therefor, those derived from E. coli K12 are known (see Eur. J. Biochem., Vol. 133, No. 1, pp. 155-162 (1983), and Biochem. J., Vol. 287, pp. 611-619, particularly p. 616, right column, lines 19-31 (1992)).
The object of the present invention is to provide a large amount of variant E.sub.1 protein of higher activity by means of genetic engineering.
In the process of cloning an E.sub.1 protein gene derived from the wild-type E. coli strain 1100 by the PCR technique, the present inventors have obtained variant E.sub.1 protein in which one amino acid is replaced by another amino acid, and they have unexpectedly found that said variant E.sub.1 protein possesses an extremely higher activity compared with the wild-type E.sub.1 protein of known sequence.